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Abstract Background TDP-43 proteinopathies are characterized by loss of nuclear TDP-43 expression and formation of C-terminal TDP-43 fragmentation and accumulation in the cytoplasm. Recent studies have shown that TDP-43 can accumulate in RNA stress granules (SGs) in response to cell stresses and this could be associated with subsequent formation of TDP-43 ubiquinated protein aggregates. However, the initial mechanisms controlling endogenous TDP-43 accumulation in SGs during chronic disease are not understood. In this study we investigated the mechanism of TDP-43 processing and accumulation in SGs in SH-SY5Y neuronal-like cells exposed to chronic oxidative stress. Cell cultures were treated overnight with the mitochondrial inhibitor paraquat and examined for TDP-43 and SG processing. Results We found that mild stress induced by paraquat led to formation of TDP-43 and HuR-positive SGs, a proportion of which were ubiquitinated. The co-localization of TDP-43 with SGs could be fully prevented by inhibition of c-Jun N-terminal kinase (JNK). JNK inhibition did not prevent formation of HuR-positive SGs and did not prevent diffuse TDP-43 accumulation in the cytosol. In contrast, ERK or p38 inhibition prevented formation of both TDP-43 and HuR-positive SGs. JNK inhibition also inhibited TDP-43 SG localization in cells acutely treated with sodium arsenite and reduced the number of aggregates per cell in cultures transfected with C-terminal TDP-43 162-414 and 219-414 constructs. Conclusions Our studies are the first to demonstrate a critical role for kinase control of TDP-43 accumulation in SGs and may have important implications for development of treatments for FTD and ALS, targeting cell signal pathway control of TDP-43 aggregation.

Positive allosteric modulator (PAM) drugs enhance the activation of the calcium-sensing receptor (CaSR) and suppress parathyroid hormone (PTH) secretion. Unfortunately, these hyperparathyroidism-treating drugs can induce hypocalcemia and arrhythmias. Seeking improved modulators, we docked libraries of 2.7 million and 1.2 billion molecules against the CaSR structure. The billion-molecule docking found PAMs with a 2.7-fold higher hit rate than the million-molecule library, with hits up to 37-fold more potent. Structure-based optimization led to nanomolar leads. In ex vivo organ assays, one of these PAMs was 100-fold more potent than the standard of care, cinacalcet, and reduced serum PTH levels in mice without the hypocalcemia typical of CaSR drugs. As determined from cryo-electron microscopy structures, the PAMs identified here promote CaSR conformations that more closely resemble the activated state than those induced by the established drugs.

Drugs acting as positive allosteric modulators (PAMs) to enhance the activation of the calcium sensing receptor (CaSR) and to suppress parathyroid hormone (PTH) secretion can treat hyperparathyroidism but suffer from side effects including hypocalcemia and arrhythmias. Seeking new CaSR modulators, we docked libraries of 2.7 million and 1.2 billion molecules against transforming pockets in the active-state receptor dimer structure. Consistent with simulations suggesting that docking improves with library size, billion-molecule docking found new PAMs with a hit rate that was 2.7-fold higher than the million-molecule library and with hits up to 37-fold more potent. Structure-based optimization of ligands from both campaigns led to nanomolar leads, one of which was advanced to animal testing. This PAM displays 100-fold the potency of the standard of care, cinacalcet, in ex vivo organ assays, and reduces serum PTH levels in mice by up to 80% without the hypocalcemia typical of CaSR drugs. Cryo-EM structures with the new PAMs show that they induce residue rearrangements in the binding pockets and promote CaSR dimer conformations that are closer to the G-protein coupled state compared to established drugs. These findings highlight the promise of large library docking for therapeutic leads, especially when combined with experimental structure determination and mechanism., Competing Interests: Competing Interests: BKS is a founder of Epiodyne, Inc, BlueDolphin, LLC, and Deep Apple Therapeutics, Inc., serves on the SAB of Schrodinger LLC and of Vilya Therapeutics, on the SRB of Genentech, and consults for Levator Therapeutics and Hyku Therapeutics. GS is a founder and consultant of Deep Apple Therapeutics, Inc. JJI co-founded Deep Apple Therapeutics, Inc., and BlueDolphin, LLC.

Introduction: Few studies have evaluated the presence of Post COVID-19 conditions (PCC) in people from Latin America, a region that has been heavily afflicted by the COVID-19 pandemic. In this study, we describe the frequency, co-occurrence, predictors, and duration of 23 symptoms in a cohort of Mexican patients with PCC., Methods: We prospectively enrolled and followed adult patients hospitalized for severe COVID-19 at a tertiary care centre in Mexico City. The incidence of PCC symptoms was determined using questionnaires. Unsupervised clustering of PCC symptom co-occurrence and Kaplan-Meier analyses of symptom persistence were performed. The effect of baseline clinical characteristics was evaluated using Cox regression models and reported with hazard ratios (HR)., Results: We found that amongst 192 patients with PCC, respiratory problems were the most prevalent and commonly co-occurred with functional activity impairment. 56% had ≥5 persistent symptoms. Symptom persistence probability at 360 days 0.78. Prior SARS-CoV-2 vaccination and infection during the Delta variant wave were associated with a shorter duration of PCC. Male sex was associated with a shorter duration of functional activity impairment and respiratory symptoms. Hypertension and diabetes were associated with a longer duration of functional impairment. Previous vaccination accelerated PCC recovery., Discussion: In our cohort, PCC symptoms were frequent (particularly respiratory and neurocognitive ones) and persistent. Importantly, prior SARS-CoV-2 vaccination resulted in a shorter duration of PCC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Núñez, Gillard, Fragoso-Saavedra, Feyaerts, Islas-Weinstein, Gallegos-Guzmán, Valente-García, Meyerowitz, Kelly, Chen, Ganio, Benkendorff, Flores-Gouyonnet, Dammann-Beltrán, Heredia-González, Rangel-Gutiérrez, Blish, Nadeau, Nolan, Crispín, McIlwain, Gaudillière and Valdés-Ferrer.)

Natural Killer (NK) cells play a key role in controlling HIV replication, with potential downstream impact on the size of the HIV reservoir and likelihood of viral rebound after antiretroviral therapy (ART) cessation. It is therefore important to understand how primary HIV infection (PHI) disrupts NK cell function, and how these functions are restored by early ART. We examined the impact of commencing ART during PHI on phenotypic and functional NK cell markers at treatment initiation (baseline), 3 months, 1 year, and 2 years in seven well-characterised participants in comparison to HIV seronegative volunteers. We then examined how those NK cell properties differentially impacted by ART related to time to viral rebound and HIV DNA levels in 44 individuals from the SPARTAC trial who stopped ART after 48 weeks treatment, started during PHI. NK cell markers that were significantly different between the seven people with HIV (PWH) treated for 2 years and HIV uninfected individuals included NKG2C levels in CD56 dim NK cells, Tim-3 expression in CD56 bright NK cells, IFN-γ expressed by CD56 dim NK cells after IL-12/IL-18 stimulation and the fraction of Eomes-/T-bet+ in CD56 dim and CD56 bright NK cells. When exploring time to viral rebound after stopping ART among the 44 SPARTAC participants, no single NK phenotypic marker correlated with control. Higher levels of IL-12/IL-18 mediated NK cell degranulation at baseline were associated with longer times to viral rebound after treatment interruption (P=0.028). Additionally, we found higher fractions of CD56 dim NK cells in individuals with lower levels of HIV DNA (P=0.048). NKG2A and NKp30 levels in CD56 neg NK cells were higher in patients with lower HIV DNA levels (p=0.00174, r=-0.49 and p=0.03, r= -0.327, respectively) while CD27 levels were higher in those with higher levels of HIV DNA (p=0.026). These data show NK cell functions are heterogeneously impacted by HIV infection with a mixed picture of resolution on ART, and that while NK cells may affect HIV DNA levels and time to viral rebound, no single NK cell marker defined delayed viral rebound., Competing Interests: Author JH was employed by Etcembly Ltd, Harwell Campus, United Kingdom. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pace, Ogbe, Hurst, Robinson, Meyerowitz, Olejniczak, Thornhill, Jones, Waters, Lwanga, Kuldanek, Hall, Zacharopoulou, Martin, Brown, Nwokolo, Peppa, Fox, Fidler and Frater.)

The μ-opioid receptor (μOR) is the major target for opioid analgesics. Activation of μOR initiates signaling through G protein pathways as well as through β-arrestin recruitment. μOR agonists that are biased towards G protein signaling pathways demonstrate diminished side effects. PZM21, discovered by computational docking, is a G protein biased μOR agonist. Here we report the cryoEM structure of PZM21 bound μOR in complex with G i protein. Structure-based evolution led to multiple PZM21 analogs with more pronounced G i protein bias and increased lipophilicity to improve CNS penetration. Among them, FH210 shows extremely low potency and efficacy for arrestin recruitment. We further determined the cryoEM structure of FH210 bound to μOR in complex with G i protein and confirmed its expected binding pose. The structural and pharmacological studies reveal a potential mechanism to reduce β-arrestin recruitment by the μOR, and hold promise for developing next-generation analgesics with fewer adverse effects., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background and Purpose: The intranasal administration of oxytocin (OT) reduces migraine headaches through activation of the oxytocin receptor (OTR). Magnesium ion (Mg 2+) concentration is critical to the activation of the OTR, and a low serum Mg 2+ concentration is predictive of a migraine headache. We, therefore, examined the functional impact of Mg 2+ concentration on OT-OTR binding efficacy using two complimentary bioassays., Experimental Approach: Current clamp recordings of rat trigeminal ganglia (TG) neurons measured the impact of Mg 2+ on an OT-induced reduction in excitability. In addition, we assessed the impact of Mg 2+ on intranasal OT-induced craniofacial analgesia in rats., Key Results: While OT alone dose-dependently hyperpolarized TG neurons, decreasing their excitability, the addition of 1.75 mM Mg 2+ significantly enhanced this effect. Similarly, while the intranasal application of OT produced dose-dependent craniofacial analgesia, Mg 2+ significantly enhanced these effects., Conclusions and Implications: OT efficacy may be limited by low ambient Mg 2+ levels. The addition of Mg 2+ to OT formulations may improve its efficacy in reducing headache pain as well as for other OT-dependent processes.

Human MAIT cells sit at the interface between innate and adaptive immunity, are polyfunctional and are capable of killing pathogen infected cells via recognition of the Class IB molecule MR1. MAIT cells have recently been shown to possess an antiviral protective role in vivo and we therefore sought to explore this in relation to HIV-1 infection. There was marked activation of MAIT cells in vivo in HIV-1-infected individuals, which decreased following ART. Stimulation of THP1 monocytes with R5 tropic HIV BAL potently activated MAIT cells in vitro. This activation was dependent on IL-12 and IL-18 but was independent of the TCR. Upon activation, MAIT cells were able to upregulate granzyme B, IFNγ and HIV-1 restriction factors CCL3, 4, and 5. Restriction factors produced by MAIT cells inhibited HIV-1 infection of primary PBMCs and immortalized target cells in vitro. These data reveal MAIT cells to be an additional T cell population responding to HIV-1, with a potentially important role in controlling viral replication at mucosal sites., Competing Interests: CP, PP, CH, JT, CM, JM, LM, CV, DG, LD, RM, QS, JM, DF, SF, AK, JF, PK No competing interests declared, (© 2021, Phetsouphanh et al.)

T cell dysfunction occurs early following HIV infection, impacting the emergence of non-AIDS morbidities and limiting curative efforts. ART initiated during primary HIV infection (PHI) can reverse this dysfunction, but the extent of recovery is unknown. We studied 66 HIV-infected individuals treated from early PHI with up to three years of ART. Compared with HIV-uninfected controls, CD4 and CD8 T cells from early HIV infection were characterised by T cell activation and increased expression of the immune checkpoint receptors (ICRs) PD1, Tim-3 and TIGIT. Three years of ART lead to partial - but not complete - normalisation of ICR expression, the dynamics of which varied for individual ICRs. For HIV-specific cells, epigenetic profiling of tetramer-sorted CD8 T cells revealed that epigenetic features of exhaustion typically seen in chronic HIV infection were already present early in PHI, and that ART initiation during PHI resulted in only a partial shift of the epigenome to one with more favourable memory characteristics. These findings suggest that although ART initiation during PHI results in significant immune reconstitution, there may be only partial resolution of HIV-related phenotypic and epigenetic changes., Competing Interests: WH is an employee of Merck and Company and holds equity in Tango Therapeutics and Arsenal Biosciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Martin, Sen, Pace, Robinson, Meyerowitz, Adland, Thornhill, Jones, Ogbe, Parolini, Olejniczak, Zacharopoulou, Brown, Willberg, Nwokolo, Fox, Fidler, Haining and Frater.)